This invention relates to methods for improving detection of various physiologically active substances in tested fluids
Immunoassays, hybridization, ligand-receptor and enzymatic assays are widely used in biology and medicine for detection of viruses, bacteria, cancer markers, and for drug screening (Edberg, 1985; Catty, 1989).
In particular, immunoassay and hybridization assays are used for detection of a number of pathogenic bacteria and viruses that can be considered possible biological warfare agents. Among them are an Anthrax (Bacillus anthracis), an Arenaviruse, a Clostridium botulinum toxin, a Brucella specie, a Burkholderia pseudomallei, a Chlamydia psittaci, a Vibrio cholerae, an Ebola virus, a variola major, a Staphylococcal enterotoxin B, a Francisella tularensis, a Rickettsia prowazekii, a Yersinia pestis, a Cryptosporidium parvum, and food- and water-borne bacteria, such as Salmonella sp., an Escherichia coli O157:H7, Shigella, Staphylococcus aureus, Campylobacter jejuni, Campylobacter coli, Listeria. monocytogenes and Bacillus cereus. They are included in bioterrorism pathogens list published by the Centers of Disease Control and Prevention (CDC). These agents are considered possible biological warfare agents because they are resistant to environmental conditions, most of the human population is completely susceptible, and the diseases some of them cause are severe with a high fatality rate. A large quantity of these organisms could easily be grown and preserved for several years. These agents are also environmental and food chain-safety threats.
The effective testing of, for instance, pathogenic bacteria requires methods of analysis that meet a number of challenging criteria. Time and sensitivity of analysis are the most important limitations related to the usefulness of microbiological testing. Bacterial detection methods have to be rapid and very sensitive since the presence of even a single pathogenic organism in the water, body or food may be an infectious dose. At present, three groups of tests for food- and water-borne bacteria are, mainly, used: 1) culture methods, 2) immunoassay, and 3) polymerase chain reaction (PCR)-based assays. Culture methods are laborious, time-consuming and very expensive. They require a minimum of several days to perform. Polymerase chain reaction (PCR) method is based on amplification of small quantities of genetic material to determine the presence of bacteria. The PCR method is rather sensitive, but requires pure samples and hours of processing and expertise in molecular biology (Meng et al., 1996; Sperveslage et al., 1996). Immunoassay methods, which yield a presumptive positive or negative screening result in 24 h or less have also been developed. Typical sensitivities of immunoassays are approximately 106 CFU/ml, although more sensitive variants of immunoassay (with sensitivities of 102-105 CFU/ml) are being developed (Yu and Bruno, 1996; Mazenko et al., 1999).
Thus, there is a need in highly sensitive, rapid and reliable analytical methods for detection of water- and food-borne pathogens. In response to this problem, considerable effort is now directed towards the development of methods that can rapidly detect low concentrations of pathogens in water, food and clinical samples. For this purpose, a number of instruments are being developed using various principles of detection, e.g. chromatography, infrared or fluorescence spectroscopy, bioluminescence, flow cytometry, impedimetry and many others (Ivnitski et al., 1999). Despite these efforts, the only a few biosensors for bacterial detection are commercially available or are approaching commercialization. The main reasons for this are the challenges to create biosensors with the necessary sensitivity and properties for reliable and effective use in routine applications. Since the present invention allows one to increase the sensitivity of the analytical biochemical methods significantly, it has a paramount value for a more reliable detection of potential biological warfare agents.
Very sensitive analytical biochemical methods are also of significant value for detection of infectious diseases. For example, in spite of using sensitive Enzyme-linked immunosorbent assays (ELISA) for the detection of HIV, Hepatits B and C viruses in blood donors, the residual risk of post-transfusion infection remains. The enzyme immunoassay testing currently used for HIV and Hepatitis B testing produces many false negative results. For example, deaths due to fulminant post-transfusion hepatitis B have been reported although HBsAg-negative blood on ELISA was transfused (Arababadi et al., 2011). The amount of HBsAg contained in the peripheral blood of these HBsAg-negative HBV carriers is so small that the currently employed methods are inadequate (Sawke and Sawke, 2010).
Other physiologically active substances that require high sensitivity methods to be detected are pathogenic bacteria, viruses, cancer biomarkers such as Clostridium difficile toxin A, a Clostridium difficile toxin B, a rotavirus, a p50 recombinant protein NFkB p50 homodimer, an RNA, a DNA, an mRNA, an cDNA, and a prostatic specific antigen (PSA).
Formats of Analytical Biochemical Assays
There are a number of various formats of immuno- and hybridization assay and ligand-receptor assays that are used in clinical and research practice. They are based on the use of either antigen-antibody, ligand-receptor or oligonucleotides hybridization reactions for detection of the analyte to be detected, and use of labels (markers) of different origin: photochemical, radioactive, enzymatic, fluorescent, chemiluminescent and others.
1. Assays Based on the Use of Photochemical Labels
It has been reported that certain dyes, for instance derivatives of fluorescein, rhodamine, erythrosine, eosin, methylene blue, Bengal rose, porphyrin, phthalocyanine and many others were used as photochemical labels in various analytical biochemical assays. These dyes possess a high yield to their triplet state, and therefore, can be used as photosensitizers (photocatalysts) of the photochemical reactions. Under certain conditions photochemical reaction between such dye and selected substrate occurs and takes place only under irradiation with certain range of light wavelengths.
Photochemical Reactions and Photooxidation.
Chemical reactions caused by absorption of light are defined as photochemical reactions. Photoexcitation is the first step in a photochemical process in which the photon can be absorbed directly by the reactant or by a photosensitizer which absorbs the photon and transfers the energy to the reactant. There are several types of photochemical reactions. One such type is photosensitized oxidation implicated in numerous processes in vivo and in vitro. Many of these oxidation processes can occur in the dark; however, light can accelerate oxidation due to the photochemical generation of free radicals (type I photosensitized oxidation) or singlet oxygen (type II photosensitized oxidation). Strongly light-absorbing organic dye molecules such as Rose Bengal, erythrosine, eosin, methylene blue, porphyrines and phthalocyanines are typical examples of photosensitizers that can participate in Types I and II photochemical reactions. In Type II photochemical reaction, produced singlet oxygen reacts with many organic compounds including aromatics, vitamins, steroids, fatty acids, aminoacids, proteins, nucleic acids, and synthetic polymers (Timoshenko, 2009). Photosensitized oxidation involving singlet oxygen is implicated in analytical assays, phototherapy of cancer, photocarcinogenesis, photodynamic inactivation of viruses and cells, and in photodegradation of organic compounds (Schmidt, 2006b; Schmidt, 2006a).
Various formats of analytical assays using photosensitizers as labels for determination of physiologically active substances (analytes) have been described in patent and scientific literature. In references (Motsenbocker et al., 1993a; Motsenbocker et al., 1993b) derivatives of methylene blue were used as labels in Enzyme-Linked Immunosorbent Assay (ELISA)-type assays. In this assay, a methylene blue dye derivative was synthesized and covalently attached to detection antibody. Upon irradiation of the solution containing luminol by pulsed red light, a photosensitive dye is excited and plays a role of photosensitizer (photocatalyst) of luminol oxidation which results in generation of blue light. An alpha-fetoprotein immunoassay based on this principle was developed having a detection limit of 17 pg.
It has also been reported that fluorescein, rodamine and eosin can be utilized as photochemical antibody labels in immunohistochemistry. It was shown that DAB can be photooxidized by these dyes in immunolabeled cultured cells (Sandell and Masland, 1988). In this case, the fluorochrome which is a label for the antibody bound to the cell, is utilized as a photosensitizer in the photochemical reaction of DAB oxidation. Since the production of reactive oxygen species by the fluorophore has been implicated in the photooxidation reaction (Sandell and Masland, 1988), the experiment made use of test fluorescent compounds that were more potent generators of singlet oxygen. Many of the compounds currently used for immunofluorescent and tyramine-based labeling, such as fluorescein and rhodamine were chosen because of their high fluorescence quantum yields, and have comparatively low yields of singlet oxygen (1O2). Eosin, a brominated derivative of fluorescein, has a singlet oxygen quantum yield (0.57) approximately 19 times greater than fluorescein (Gandin et al., 1983), while still possessing moderate fluorescence (˜20% as bright as fluorescein) (Fleming et al., 1977). Other fluorescein derivatives such as erythrosine and Rose Bengal are also known to be effective photosensitizers and singlet oxygen generators, and can be used as labels in immunohistochemistry.
Two homogeneous (not requiring physical separation of labeled and unlabeled reagents) immunoassay techniques based on the use of photogeneration of singlet oxygen have been independently developed. The first method is based on (a) photooxidation by singlet oxygen (1O2) of a fluorescent substrate (1,3-diphenylisobenzofuran, DPBF) embedded in unilamellar vesicles on the surface of which antibody to the analyte antigen is covalently attached (DPBF-immunoliposomes); (b) generation of singlet oxygen, upon illumination, by a chromophore (erythrosine) covalently attached to an antibody (Ab*) or antigen (Ag*); (c) formation of a “sandwich”- or “competition”-type complex whereupon the singlet oxygen-generating chromophore conjugate (Ab* or Ag*) and immunoliposome-embedded DPBF are brought into close proximity (Bystryak et al., 1995; Bystryak, 1998). Competition- and sandwich-type model assay systems for the detection of protein antigens and viruses were developed.
In the second method, the Luminescent Oxygen Channeling Immunoassay (LOCI) (Ullman et al., 1994), singlet oxygen is generated by a photosensitizer and an antenna dye that are dissolved in one of the particles. Singlet oxygen molecule diffuses to the second particle and initiates a chemiluminescent reaction of an olefin that is dissolved in it. The technique permits real-time measurement of particle binding kinetics when analyte is present in the solution. By using antibody-coated particles, a homogeneous immunoassay capable of detecting approximate to 4 amol of thyroid-stimulating hormone in 12 min was demonstrated.
AlphaScreen/AlphaLISA assays developed based upon an oxygen channeling technology, LOCI are bead based proximity assays when the donor, which contains phthalocyanine, is laser excited and ambient oxygen is converted to singlet oxygen. This is a highly amplified reaction since approximately 60,000 singlet oxygen molecules can be generated and travel at least 200 nm in aqueous solution before decay. Consequently, if the Donor and Acceptor beads are within that proximity, energy transfer occurs. Singlet oxygen reacts with chemicals (substrates) in the Acceptor beads to produce a luminescent response (Eglen et al., 2008). AlphaScreen/AlphaLISA assays are immunoassays as well as high throughput drug screening assays involving antigen-antibody, oligonucleotide hybridization, biotin-streptavidin, ligand-receptor binding reactions and enzymatic reactions.
2. Assays Using Enzymes as Labels
Since the introduction, in 1966, of enzymes as markers for the labeling of antigens and antibodies (Avrameas and Uriel, 1966; Nakane and Pierce, 1966), immunoenzymatic techniques have been considerably developed and diversified. These techniques are now routinely used for detection of physiologically active substances in body fluids, localization of antigens or antibodies on tissues, detection of antigens or antibodies immobilized on various solid phases, as well as for the titration of antibodies, and for the precise measurement of antigens. Antigens and/or antibodies are localized, detected and/or titrated by means of various heterogeneous procedures.
In general, heterogeneous immunoenzymatic procedures are based on the use and detection of enzyme-antibody or enzyme-antigen conjugates, prepared according to several established protocols. Sometimes, antibody or antigen is coupled to enzyme through biotin-streptavidin bond, which is antibody (or antigen)-biotin-streptavidin-enzyme conjugate is used. The enzyme is detected using chromogenic, fluorogenic or chemiluminescent enzyme substrates by detecting a corresponding signal obtained from the product of the enzymatic reaction.
For example, in conventional Enzyme Linked Immuno-Sorbent Assay (ELISA), antibody against analyte to be detected is attached to solid phase, particularly, polystyrene microtiter plate. Then, body fluid with unknown concentration of the analyte and enzyme-labeled antibody to analyte are added. This results in formation of antibody-analyte-enzyme-labeled antibody complex on the surface of the solid phase. After incubation, the unbound analyte and enzyme-labeled antibody is removed from the solution by rinsing. Then, a substrate solution is added to the test tube or well, and the amount of the bound enzyme-conjugated antibody and, consequently, the concentration of the analyte is determined by detecting color, fluorescence or luminescence signals of the liquid phase in the final reaction mixture.
Other than ELISA methods that use enzyme as a label include hybridization assays, Immunohistochemistry (IHC) and in situ hybridization (ISH) assays, blotting analysis, immunochromatography and other methods. Hybridization and in situ hybridization (ISH) assays are based on oligonucleotides binding whereas Immunohistochemistry (IHC), blotting analysis and immunochromatography methods are based on antigen-antibody reactions in the same way as ELISA.
However, in many cases, the sensitivity of these assays is inadequate. Therefore, there is a need in development of more sensitive assays. In order to detect constituents (analytes) present in small amounts, it is necessary to devise procedures capable of strongly amplifying the signal and/or increasing of signal-to-noise ratio, which results in increasing of the analytical sensitivity of the assay. Since all heterogeneous immunoenzymatic techniques involve, in their final step, the detection of an enzyme associated with solid phase, essentially two approaches have been developed to obtain such enzymatic signal amplification. The first includes procedures leading to a high accumulation of enzyme labels associated with the solid phase. The second consists of procedures that make use of enzyme substrate derivatives, which give rise to reaction products detectable in minute amounts. The present invention relates to the second approach, which is also applicable to some assays using photosensitized photochemical reactions.
A type of amplification technique that can be employed for enzyme-mediated assays utilizes light or photonic energy to increase the sensitivity of the assay. This technique is disclosed in U.S. Pat. No. 5,776,703. It is widely known that some chemical reactions are photosensitive dependent upon the quantum chemical structure and other properties of the reactants. The rate of a photosensitive reaction is much higher if the reaction mixture is illuminated by an intense light of a specific wavelength compared to a similar reaction taking place in the absence of such light.
The technique described in the '703 patent includes the binding of an antibody to a suspected antigen, wherein the antibody is labeled with an enzyme such as HRP and added to a biological liquid, for example blood or serum. A portion of the HRP-labeled antibodies binds with the antibodies that are specific to the antigen and existing already in the biological liquid to form an [antibody]-[antigen]-[HRP labeled antibody] complex. Subsequently, after an incubation time, the HRP-labeled antibody which did not bind to the Ab-Ag complex is removed from the solution by rinsing or washing. Then, a substrate solution containing H2O2 and OPD (orthophenylenediamine) is added to the test tube and the OPD is oxidized with HRP acting as an oxidizing catalyst. The oxidation product of HRP-catalyzed reaction of OPD oxidation is diaminophenazine (DAP). DAP is a colored substance, and the optical density of the solution containing DAP can be read with the aid of a spectrophotometer or microplate reader. At this point in the assay a stopping solution, such as sulfuric acid, is used in order to stop the various chemical reactions from proceeding and producing reaction products that may interfere with an accurate measurement. The final signal is proportional to the concentration of HRP bound to antibodies and therefore to the added antibodies forming the Ab-Ag-(Ab-HRP) complex.
The '703 patent further discloses that the procedure performed to this point can be enhanced by the application of intense light at the wavelength of 400 to 500 nm before adding a stop solution containing acid. Prior to a spectrophotometer (or fluorometer) reading, the test tube is illuminated by an intense source of light of a wavelength in the above range. The DAP obtained in the first stage of the reaction is a photosensitizer for the following photochemical reaction of OPD oxidation, and together with the light photons serve as new catalyzing agents for further production of DAP. Thus a two stage process takes place:

The resulting optical density or fluorescence of the sample is measured by a spectrophotometer or fluorometer, respectively, and the results obtained by the '703 patent are an improvement over the prior art. However, this method suffers from the limited sensitivity due to the fact that both useful and background (noise) signals increase within a photochemical step (1.2). This severely restricts the utility of the method. It should be emphasized that other binding agents and pairs such as oligonucleotides, oligonucleotide-protein, biotin-avidin(streptavidin) and protein-protein can be used in such kind of assays.
An attempt of modification of the technique disclosed in U.S. Pat. No. 5,776,703 was done in US PATENT APPLICATION PUBLICATION No. 2002/0110842, which is incorporated herein by reference in its entirety. The '842 application describes adding of some detergents to the OPD substrate solution to increase the sensitivity of the assay including a photochemical amplification step. The best mode of practicing the '842 invention included the commercially available detergent Triton X-100 as an additive at a concentration in the range of 10% in a phosphate/citrate buffer with a pH of approximately 5.0. Triton X-100 has a chemical formula of C14H22O(C2H4O)n, where the average number of ethylene oxide units per molecule, n, ranges from 9 to 10.
Also disclosed is the use of Tween-20, another commercially available detergent, as an additive. However, the addition of detergents to the OPD substrate solution results in increase of the rate of the photochemical reaction (1.2) only; the sensitivity of the method was not improved as compared to the method described in the '703 patent.
Although the methods described in '703 patent and '842 application have increased the sensitivities of assays versus prior art methods, it is still desirable to have an even more sensitive technique. In addition to sensitivity, it would be beneficial to have an immunoassay method that further improved the signal-to-noise (S/N) ratio, which would substantially increase the effective range of the known methods. The methods of the '703 patent and the '842 application are adversely affected to a large degree by background noise. In addition, these methods are highly dependent upon a number of factors including: (1) the wavelength of the light used to catalyze the reaction; (2) the intensity of the light (the number of photons used to catalyze the reaction); and, (3) the illumination exposure (intensity x time).
It is therefore desirable to have an assay of detecting an analyte in a sample with increased sensitivity compared to known assays. It is also desirable to have an assay for detecting an analyte in a sample with an improved S/N ratio compared to known assays. It is also desirable to have an assay for detecting an analyte in a sample that can be used with existing ELISA and other assays methodology.